A number of monoclonal antibodies after immunization with human tumor cells have been selected by positive reactivity with human cancer cell lines and negative reactivity with normal tissues and normal cell lines. Many of these antibodies have been identified as being directed to mucin-type glycoproteins, which are characterized by extremely heterogeneous disulfide interlinked high molecular weight polypeptides having a large number of O-linked oligosaccharides. The majority of O-linked oligosaccharides consist of a disaccharide core (Gal.beta.1.fwdarw.3GalNAc.alpha.1.fwdarw.O-Ser/Thr) to which a number of sugar residues such as sialic acid, fucose, and N-acetyl glucosamine are linked to form highly complex structures in the human species. These mucin-type glycoproteins are mainly present in epithelial cells and are secreted from goblet cells to form mucinous secretions.
Associated with oncogenic transformation, synthesis of many sugar chains is blocked (Hakomori & Murakami, Proc. Natl. Acad. Sci. USA, 59:254-261, 1968 and Hakomori, Cancer Res., 45:2405-2414, 1985). Synthesis of carbohydrate chains in mucin-type glycoproteins is also blocked strongly in many human cancers, thus forming a number of mucin-type glycoproteins with short carbohydrate chains and without peripheral structures, i.e., without modification of the core structure. Among those glycoproteins with incomplete oligosaccharide chains, T, Tn, and sialyl-Tn have been identified. These are the common core structures of all mucin-type glycoproteins present in normal tissues in a cryptic form (Springer, Science, 224:1198-1206, 1984 and Hirohashi et al., Proc. Natl. Acad. Sci. USA, 82:7039-7043, 1985).
The current methods used to prepare monoclonal antibodies to human cancer-associated antigens use as the immunogen tumor tissue homogenates or tumor cell lines, and the hybridoma is selected by analyzing the specificity of the antibodies produced with a large number of tumor cells and tissue sections.
For example, the blood group A cross-reacting antigenicity of an .alpha.-GalNAc residue linked to a serine or threonine of a glycoprotein has been described by Uhlenbruck and Associates as the Tn antigen detectable by various GalNAc lectins (Helix pomatia, Soja hispida, and Sarothamnus scoparius) that preferentially agglutinate blood group A erythrocytes. The epitope represents the innermost .alpha.-GalNAc residue of O-linked carbohydrate chains present in transmembrane glycoproteins as well as mucin-type glycoproteins. Therefore, the antigen is cryptic in normal cells or secretions, but becomes exposed after desialylation followed by Smith degradation (Dahr et al., Vox Sang., 27:29-42, 1974). The Tn antigen has also been described by Springer and Associates as the precursor of the Thomsen-Friedenreich antigen (T antigen), and both Tn and T antigens are expressed in breast cancer (Springer et al., J. Natl. Cancer Inst., 54:335, 1975) and various other cancers (Springer et al., Cancer, 55:561-569, 1985). However, the wide occurrence of the antigen in various types of human cancer and its restricted distribution in normal tissues were not well recognized until monoclonal antibodies specific to this epitope were recently established (Hirohashi et al., Proc. Natl. Acad. Sci. USA, 82:7039-7043, 1985). The IgM antibodies NCC-LU-35 and NCC-LU-81 were originally established after immunization of mice with human lung squamous cell carcinoma Lu65 and selected by specific reactivity with tumors but not with normal tissues. These antibodies were shown to cross-react with A antigen and were identified as being directed to the Tn antigen (Hirohashi et al., Proc. Natl. Acad. Sci. USA, 82:7039-7043, 1985). Although a great deal of cross-reactivity with blood group A antigen was demonstrated for NCC-LU-35 and to a lesser extent for NCC-LU-81, these antibodies are capable of defining Tn antigen specifically in tumors of blood group O and B individuals.
Since the reactivity of these anti-Tn antibodies was found to be relatively specific for non-A tumor tissues (Hirohashi et al., Proc. Natl. Acad. Sci. USA, 82:7039-7043, 1985), it would be highly desirable to obtain the IgG version of the anti-Tn antibody without cross-reactivity to blood group A, since only IgG antibodies are capable of provoking antibody dependent cytotoxic effects on tumor cells (Young & Hakamori, Science, 211:487-489, 1981; Houghton et al., Proc. Natl. Acad. Sci. USA, 82:1242-1246, 1985; Herberman et al., in Reisfeld & Sell, eds., Monoclonal Antibodies and Cancer Therapy, pp. 193-203, Alan R. Liss, Inc., New York, 1985; and Herlyn et al., Cancer Res., 40:717-721, 1980). IgG antibodies are also more suitable than IgM for preparation of conjugates with toxins, other antitumor reagents, and radioactive or paramagnetic markers for therapeutics as well as for imaging of tumors.
Further, two monoclonal antibodies, B72.3 (Nuti et al., J. Inst. Cancer, 29:539-545, 1982; Thor et al., Cancer Res., 46:3118-3124, 1986; and Johnson et al., Cancer Res., 46:850-857, 1986) and MLS102 (Kurasawa et al., in Dao et al., eds., Tumor Markers and Their Significance in the Management of Breast Cancer, p. 47-70, Alan R. Liss Inc., N.Y., 1986) have been established after immunization with human metastatic breast cancer and a colonic cancer cell line, respectively, that show highly specific reactivity with various human cancers and restricted reactivity with normal tissue.
However, these current methods require a large number of steps and, therefore, are quite cumbersome and time consuming.
Accordingly, a more efficient method for producing monoclonal antibodies to human cancer-associated antigens would be desirable as would the isolation of hybridomas that secrete monoclonal antibodies that do not exhibit undesirable cross-reactivity with glycoproteins having normal carbohydrate sequence and structure.
Further, active immunization in order to suppress tumor growth has had a long history in tumor immunology. Some successful results have been obtained in experimental tumors, but many other attempts have failed. This was partially due to the fact that the tumor antigens used were not well defined chemically, and therefore, their ability to induce host immune response to suppress tumor growth was ambiguous. Recently, a number of tumor-associated carbohydrate antigens defined by monoclonal antibodies have been well characterized chemically. The use of such antigens as immunogens to suppress tumor growth is now possible.
Chemically-defined cancer-associated glycoproteins themselves could be used to actively immunize cancer patients in order to elicit their endogenous immune response against tumors. A glycoprotein, TCA, isolated from Lewis lung carcinoma bearing T and Tn antigens was used to immunize cancer patients, which resulted in partial or complete regression of 33/71 cancer patients (Adachi et al., Anticancer Res. 4:1-4, 1984).